Oligomers

ABSTRACT

Molecules are provided for inducing or facilitating exon skipping in forming spliced mRNA products from pre-mRNA molecules in cells. The molecules may be provided directly as oligonucleotides or expression products of vectors that are administered to a subject. High rates of skipping can be achieved. High rates of skipping reduce the severity of a disease like Duchene Muscular Dystrophy so that the disease is more like Becker Muscular Dystrophy. This is a severe reduction in symptom severity and mortality.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to molecules which are capable of causingexon skipping and, in particular, relates to molecules which are capableof causing exon skipping in the dystrophin gene.

BACKGROUND OF THE INVENTION

Duchenne muscular dystrophy (DMD) is a severe X-linked muscle wastingdisease, affecting 1:3500 boys. Prognosis is poor: loss of mobility bythe age of 12, compromised respiratory and cardiac function by lateteens, and probable death by the age of 30. The disease is caused bymutations within the large dystrophin gene, such that the reading frameis disrupted leading to lack of dystrophin protein expression andbreakdown of muscle fibre integrity [1]. The dystrophin gene is large,with 79 exons. The most common DMD mutation is genomic deletion of oneor more exons, generally centred around hotspots involving exons 44 to55 and the 5′ end of the gene [2]. Mutations of the dystrophin gene thatpreserve the reading frame result in the milder, non-life threateningBecker muscular dystrophy (BMD).

Exon skipping induced by antisense oligoribonucleotides (AOs), generallybased on an RNA backbone, is a future hope as a therapy for DMD in whichthe effects of mutations in the dystrophin gene can be modulated througha process of targeted exon skipping during the splicing process. Thesplicing process is directed by complex multi-particle machinery thatbrings adjacent exon-intron junctions in pre-mRNA into close proximityand performs cleavage of phosphodiester bonds at the ends of the intronswith their subsequent reformation between exons that are to be splicedtogether. This complex and highly precise process is mediated bysequence motifs in the pre-mRNA that are relatively short semi-conservedRNA segments to which bind the various nuclear splicing factors that arethen involved in the splicing reactions. By changing the way thesplicing machinery reads or recognises the motifs involved in pre-mRNAprocessing, it is possible to create differentially spliced mRNAmolecules. It has now been recognised that the majority of human genesare alternatively spliced during normal gene expression, although themechanisms involved have not been identified. Using antisenseoligonucleotides, it has been shown that errors and deficiencies in acoded mRNA could be bypassed or removed from the mature genetranscripts. Indeed, by skipping out-of-frame mutations of thedystrophin gene, the reading frame can be restored and a truncated, yetfunctional, Becker-like dystrophin protein is expressed. Studies inhuman cells in vitro [3, 4] and in animal models of the disease in vivo[5-9] have proven the principle of exon skipping as a potential therapyfor DMD (reviewed in [10]). Initial clinical trials using two differentAO chemistries (phosphorodiamidate morpholino oligomer (PMO) andphosphorothioate-linked 2′-O-methyl RNA (2′OMePS)) [11] have recentlybeen performed, with encouraging results. Indisputably impressiverestoration of dystrophin expression in the TA muscle of four DMDpatients injected with a 2′OMePS AO to exon 51 has been reported by vanDeutekom et al. [11].

However, it should be noted that, relative to 2′OMePS AOs, PMOs havebeen shown to produce more consistent and sustained exon skipping in themdx mouse model of DMD [12-14; A. Malerba et al, manuscript submitted],in human muscle explants [15], and in dystrophic canine cells in vitro[16]. Most importantly, PMOs have excellent safety profiles fromclinical and pre-clinical data [17].

The first step to a clinical trial is the choice of the optimal AOtarget site for skipping of those dystrophin exons most commonly deletedin DMD. In depth analysis of arrays of 2′OMePS AOs have been reported[18, 19], and relationships between skipping bioactivity and AOvariables examined.

One problem associated with the prior art is that the antisenseoligonucleotides of the prior art do not produce efficient exonskipping. This means that a certain amount of mRNA produced in thesplicing process will contain the out-of-frame mutation which leads toprotein expression associated with DMD rather than expression of thetruncated, yet functional, Becker-like dystrophin protein associatedwith mRNA in which certain exons have been skipped.

Another problem associated with the prior art is that antisenseoligonucleotides have not been developed to all of the exons in thedystrophin gene in which mutations occur in DMD.

An aim of the present invention is to provide molecules which causeefficient exon skipping in selected exons of the dystrophin gene, thusbeing suitable for use in ameliorating the effects of DMD.

SUMMARY OF THE INVENTION

The present invention relates to molecules which can bind to pre-mRNAproduced from the dystrophin gene and cause a high degree of exonskipping in a particular exon. These molecules can be administeredtherapeutically.

The present invention provides a molecule for ameliorating DMD, themolecule comprising at least a 25 base length from a base sequenceselected from:

(SEQ ID NO: 1) a) XGA AAA CGC CGC CAX XXC XCA ACA GAX CXG;(SEQ ID NO: 2) b) CAX AAX GAA AAC GCC GCC AXX XCX CAA CAG;(SEQ ID NO: 3) c) XGX XCA GCX XCX GXX AGC CAC XGA XXA AAX;(SEQ ID NO: 4) d) CAG XXX GCC GCX GCC CAA XGC CAX CCX GGA;(SEQ ID NO: 5) e) XXG CCG CXG CCC AAX GCC AXC CXG GAG XXC;(SEQ ID NO: 6) f) XGC XGC XCX XXX CCA GGX XCA AGX GGG AXA;(SEQ ID NO: 7) g) CXX XXA GXX GCX GCX CXX XXC CAG GXX CAA;(SEQ ID NO: 8) h) CXX XXC XXX XAG XXG CXG CXC XXX XCC AGG;(SEQ ID NO: 9) i) XXA GXX GCX GCX CXX XXC CAG GXX CAA GXG;(SEQ ID NO: 10) j) CXG XXG CCX CCG GXX CXG AAG GXG XXC XXG;(SEQ ID NO: 11) k) CAA CXG XXG CCX CCG GXX CXG AAG GXG XXC; or(SEQ ID NO: 12) l) XXG CCX CCG GXX CXG AAG GXG XXC XXG XAC;wherein the molecule's base sequence can vary from the above sequence atup to two base positions, and wherein the molecule can bind to a targetsite to cause exon skipping in an exon of the dystrophin gene.

The exon of the dystrophin gene is selected from exons 44, 45, 46 or 53.More specifically, the molecule that causes skipping in exon 44comprises at least a 25 base length from a base sequence selected from:

(SEQ ID NO: 1) a) XGA AAA CGC CGC CAX XXC XCA ACA GAX CXG,(SEQ ID NO: 2) b) CAX AAX GAA AAC GCC GCC AXX XCX CAA CAG; or(SEQ ID NO: 3) c) XGX XCA GCX XCX GXX AGC CAC XGA XXA AAX,wherein the molecule's sequence can vary from the above sequence at upto two base positions, and wherein the molecule can bind to a targetsite to cause exon skipping in exon 44 of the dystrophin gene.

The molecule that causes skipping in exon 45 comprises at least a 25base length from a base sequence selected from:

(SEQ ID NO: 4) d) CAG XXX GCC GCX GCC CAA XGC CAX CCX GGA; or(SEQ ID NO: 5) e) XXG CCG CXG CCC AAX GCC AXC CXG GAG XXC,wherein the molecule's sequence can vary from the above sequence at upto two base positions, and wherein the molecule can bind to a targetsite to cause exon skipping in exon 45 of the dystrophin gene.

The molecule that causes skipping in exon 46 comprises at least a 25base length from a base sequence selected from:

(SEQ ID NO: 6) f) XGC XGC XCX XXX CCA GGX XCA AGX GGG AXA;(SEQ ID NO: 7) g) CXX XXA GXX GCX GCX CXX XXC CAG GXX CAA;(SEQ ID NO: 8) h) CXX XXC XXX XAG XXG CXG CXC XXX XCC AGG; or(SEQ ID NO: 9) i) XXA GXX GCX GCX CXX XXC CAG GXX CAA GXG,wherein the molecule's sequence can vary from the above sequence at upto two base positions, and wherein the molecule can bind to a targetsite to cause exon skipping in exon 46 of the dystrophin gene.

The molecule that causes skipping in exon 53 comprises at least a 25base length from a base sequence selected from:

(SEQ ID NO: 10) j) CXG XXG CCX CCG GXX CXG AAG GXG XXC XXG;(SEQ ID NO: 11) k) CAA CXG XXG CCX CCG GXX CXG AAG GXG XXC; or(SEQ ID NO: 12) l) XXG CCX CCG GXX CXG AAG GXG XXC XXG XAC,wherein the molecule's sequence can vary from the above sequence at upto two base positions, and wherein the molecule can bind to a targetsite to cause exon skipping in exon 53 of the dystrophin gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a scheme summarizing the tools used in the design of PMOsto exon 53. (a) Results of ESEfinder analysis, showing the location andvalues above threshold for SF2/ASF, SF2/ASF (BRCA1), SC35, SRp40 andSRp55, shown as grey and black bars, as indicated in the legend above.(b) Output of PESX analysis, showing the location of exonic splicingenhancers as solid lines, and exonic splicing silencer as a dashed line.(c) Rescue ESE analysis for exon 53, showing predicted ESEs by lines,and where they overlap, by a ladder of lines. (d) AccessMapper analysisof in vitro hybridization. Synthetic pre-mRNA (SEQ ID NO: 25) containingexon 53 and surrounding introns was subjected to a hybridization screenagainst a random hexamer oligonucleotide array, as described inMaterials and Methods. Areas of hybridization, suggestive of areas ofopen conformation, are indicated by peaks on the graph. (e) The positionof the target sites of two 2′OMePS AOs studied previously [18] are shownfor comparison. (f) The location of the target sites for all the 25 merand 30 mer PMOs to exon 53 used in this study are indicated by lines,and numbered according to the scheme used in Table 1, except forexclusion of the prefix “h53”.

FIG. 2 shows a comparison of active (effective) and inactive(ineffective) PMOs. RT-PCR analysis of mRNA from normal human skeletalmuscle cells treated with PMOs to exon 53 demonstrates a wide variationin the efficiency of exon skipping. Over 75% exon skipping is seen withh53A30/2 (lane 5) and h53A30/3 (lane 6). h53A30/1 (lane 4) producedaround 50% skipping, while the 25-mer h53A1 (lane 3) produced just over10% skipping. In contrast, h53C1 (lane 2) was completely inactive. Lane1 contains a negative control in which cells were treated withlipofectin but no PMO.

FIG. 3 shows an Mfold secondary structure prediction for exon 53 of thehuman dystrophin gene. MFOLD analysis [25] was performed using exon 53plus 50 nt of the upstream and downstream introns (SEQ ID NO: 26), andwith a maximum base-pairing distance of 100 nt. The intron and exonboundaries are indicated, as are the positions of the target sites ofthe bioactive PMO h53A30/2 (87.2% skip) and an inactive PMO (h53B2).Examples of open and closed RNA secondary structure are arrowed.

FIG. 4 shows boxplots of parameters significant to strong PMObioactivity. Comparisons were made between inactive PMOs and thoseinducing skipping at levels in excess of 75%. Boxplots are shown forparameters which are significant on a Mann-Whitney rank sum test: PMO totarget binding energy, distance of the target site from the spliceacceptor site, the percentage overlap with areas of open conformation,as predicted by MFOLD software, and the percentage overlap of the targetsite with the strongest area accessible to binding, as revealed byhexamer hybridization array analysis. Degrees of significance areindicated by asterisks. *: p<0.05; **: p<0.01; ***: p<0.001.

FIG. 5 shows boxplots of parameters significantly different betweenbioactive (effective) and inactive (ineffective) PMOs. Comparisons weremade between PMOs determined as bioactive (those that induced skippingat greater than 5%) and those that were not. Boxplots are shown forparameters which are significant from a Mann-Whitney rank sum test: PMOto target binding energy, distance of the target site from the spliceacceptor site, the score over threshold for a predicted binding site forthe SR protein SF2/ASF, and the percentage overlap of the target sitewith the strongest area accessible to binding, as revealed by hexamerhybridization array analysis. Degrees of significance are indicated byasterisks. *: p<0.05; **: p<0.01; ***: p<0.001.

FIG. 6 shows a comparison of bioactivity of PMOs targeted to exon 53 innormal hSkMCs. Myoblasts were transfected with each of the 25 mer (panela) and 30 mer (panel b) PMOs indicated at 500 nM using lipofectin (1:4).RNA was harvested after 24 hours and subjected to nested RT-PCR andproducts visualised by agarose gel electrophoresis.

FIG. 7 shows low dose efficacy and timecourse of skipping of the mostbioactive PMOs in normal hSkMCs. (a) hSkMC myoblasts were transfectedwith the PMOs indicated over a concentration range of 25 nM to 100 nMusing lipofectin (1:4). RNA was harvested after 24 hours and subjectedto nested RT-PCR, and products visualised by agarose gelelectrophoresis. (b) hSkMC myoblasts were transfected with 100 nM and500 nM concentrations of PMO-G (+30+59) using lipofectin. RNA washarvested at the timepoints indicated following transfection andsubjected to nested RT-PCR, and products visualised by agarose gelelectrophoresis. Skipped (248 bp) and unskipped (460 bp) products areshown schematically.

FIG. 8 shows blind comparison of 13 PMO oligonucleotide sequences toskip human exon 53. Myoblasts derived from a DMD patient carrying adeletion of dystrophin exons 45-52 were transfected at 300 nM induplicate with each of the PMOs by nucleofection. RNA was harvested 3days following transfection, and amplified by nested RT-PCR. (a) Barsindicate the percentage of exon skipping achieved for each PMO, derivedfrom Image J analysis of the electropherogram of the agarose gel (b).Skipped (477 bp) and unskipped (689 bp) products are shownschematically.

FIG. 9 shows the dose-response of the six best-performing PMOs. (a)Myoblasts derived from a DMD patient carrying a deletion of dystrophinexons 45-52 were transfected with the six best-performing PMOs bynucleofection, at doses ranging from 25 nM to 400 nM. RT-PCR productsderived from RNA isolated from cells 3 days post-transfection wereseparated by agarose gel electrophoresis. (b) The percentage of exonskipping observed is expressed for each concentration of each PMO as acomparison of the percentage OD of skipped and unskipped band, asmeasured using Image J.

FIG. 10 shows persistence of dystrophin expression in DMD cellsfollowing PMO treatment. (a) Myoblasts derived from a DMD patientcarrying a deletion of dystrophin exons 45-52 were transfected bynucleofection at 300 nM with each of the six best-performing PMOs, andwere cultured for 1 to 10 days before extracting RNA. The percentage ofexon skipping was compared using the percentage OD of skipped andunskipped bands, measured using Image J analysis of the agarose gel ofthe nested RT-PCR products shown in (b). The experiment was repeated,but this time using the two best-performing PMOs from the previousanalysis, and continuing the cultures for 21 days post-transfection (cand d). (e) Western blot analysis was performed on total proteinextracts from del 45-52 DMD cells 7 days after transfection with the sixbest PMOs (300 nM). Blots were probed with antibodies to dystrophin, todysferlin as a muscle-specific loading control, and protogold for totalprotein loading control. CHQSB myoblasts, after 7 days ofdifferentiation were used as a positive control for dystrophin protein(normal).

FIG. 11 shows a comparison of most active PMOs in hDMD mice. PMOs wereinjected in a blind experiment into the gastrocnemius muscle of hDMDmice. RT-PCR analysis of RNA harvested from isolated muscle (L=left,R=right) was performed and products visualised by agarose gelelectrophoresis. Quantification of PCR products was performed using aDNA LabChip.

DETAILED DESCRIPTION OF THE INVENTION

Without being restricted to any particular theory, it is thought by theinventors that the binding of the molecules to the dystrophin pre-mRNAinteracts with or interferes with the binding of SR proteins to the exonof interest. SR proteins are involved in the slicing process of adjacentexons. Therefore, it is thought that interacting or interfering with thebinding of the SR proteins interferes with the splicing machineryresulting in exon skipping.

The base “X” in the above base sequences is defined as being thymine (T)or uracil (U). The presence of either base in the sequence will stillallow the molecule to bind to the pre-mRNA of the dystrophin gene as itis a complementary sequence. Therefore, the presence of either base inthe molecule will cause exon skipping. The base sequence of the moleculemay contain all thymines, all uracils or a combination of the two. Onefactor that can determine whether X is T or U is the chemistry used toproduce the molecule. For example, if the molecule is aphosphorodiamidate morpholino oligonucleotide (PMO), X will be T as thisbase is used when producing PMOs. Alternatively, if the molecule is aphosphorothioate-linked 2′-O-methyl oligonucleotide (2′OMePS), X will beU as this base is used when producing 2′OMePSs. Preferably, the base “X”is only thymine (T).

The advantage provided by the molecule is that it causes a high level ofexon skipping. Preferably, the molecule causes an exon skipping rate ofat least 50%, more preferably, at least 60%, even more preferably, atleast 70%, more preferably still, at least 76%, more preferably, atleast 80%, even more preferably, at least 85%, more preferably still, atleast 90%, and most preferably, at least 95%.

The molecule can be any type of molecule as long as it has the selectedbase sequence and can bind to a target site of the dystrophin pre-mRNAto cause exon skipping. For example, the molecule can be anoligodeoxyribonucleotide, an oligoribonucleotide, a phosphorodiamidatemorpholino oligonucleotide (PMO) or a phosphorothioate-linked2′-O-methyl oligonucleotide (2′OMePS). Preferably, the oligonucleotideis a PMO. The advantage of a PMO is that it has excellent safetyprofiles and appears to have longer lasting effects in vivo compared to2′OMePS oligonucleotides. Preferably, the molecule is isolated so thatit is free from other compounds or contaminants.

The base sequence of the molecule can vary from the selected sequence atup to two base positions. If the base sequence does vary at twopositions, the molecule will still be able to bind to the dystrophinpre-mRNA to cause exon skipping. Preferably, the base sequence of themolecule varies from the selected sequence at one base position and,more preferably, the base sequence does not vary from the selectedsequence. The less that the base sequence of the molecule varies fromthe selected sequence, the more efficiently it binds to the specificexon region in order to cause exon skipping.

The molecule is at least 25 bases in length. Preferably, the molecule isat least 28 bases in length. Preferably, the molecule is no more than 35bases in length and, more preferably, no more than 32 bases in length.Preferably, the molecule is between 25 and 35 bases in length, morepreferably, the molecule is between 28 and 32 bases in length, even morepreferably, the molecule is between 29 and 31 bases in length, and mostpreferably, the molecule is 30 bases in length. It has been found that amolecule which is 30 bases in length causes efficient exon skipping. Ifthe molecule is longer than 35 bases in length, the specificity of thebinding to the specific exon region is reduced. If the molecule is lessthan 25 bases in length, the exon skipping efficiency is reduced.

The molecule may be conjugated to or complexed with various entities.For example, the molecule may be conjugated to or complexed with atargeting protein in order to target the molecule to muscle tissue.Alternatively, the molecule may be complexed with or conjugated to adrug or another compound for treating DMD. If the molecule is conjugatedto an entity, it may be conjugated directly or via a linker. In oneembodiment, a plurality of molecules directed to exon skipping indifferent exons may be conjugated to or complexed with a single entity.Alternatively, a plurality of molecules directed to exon skipping in thesame exon may be conjugated to or complexed with a single entity. Forexample, an arginine-rich cell penetrating peptide (CPP) can beconjugated to or complexed with the molecule. In particular,(R-Ahx-R)(4)AhxB can be used, where Ahx is 6-aminohexanoic acid and B isbeta-alanine [35], or alternatively (RXRRBR)2XB can be used [36]. Theseentities have been complexed to known dystrophin exon-skipping moleculeswhich have shown sustained skipping of dystrophin exons in vitro and invivo.

In another aspect, the present invention provides a vector forameliorating DMD, the vector encoding a molecule of the invention,wherein expression of the vector in a human cell causes the molecule tobe expressed. For example, it is possible to express antisense sequencesin the form of a gene, which can thus be delivered on a vector. One wayto do this would be to modify the sequence of a U7 snRNA gene to includean antisense sequence according to the invention. The U7 gene, completewith its own promoter sequences, can be delivered on an adeno-associatedvirus (AAV) vector, to induce bodywide exon skipping. Similar methods toachieve exon skipping, by using a vector encoding a molecule of theinvention, would be apparent to one skilled in the art.

The present invention also provides a pharmaceutical composition forameliorating DMD, the composition comprising a molecule as describedabove or a vector as described above and any pharmaceutically acceptablecarrier, adjuvant or vehicle.

Pharmaceutical compositions of this invention comprise any molecule ofthe present invention, and pharmaceutically acceptable salts, esters,salts of such esters, or any other compound which, upon administrationto a human, is capable of providing (directly or indirectly) thebiologically active molecule thereof, with any pharmaceuticallyacceptable carrier, adjuvant or vehicle. Pharmaceutically acceptablecarriers, adjuvants and vehicles that may be used in the pharmaceuticalcompositions of this invention include, but are not limited to, ionexchangers, alumina, aluminum stearate, lecithin, serum proteins, suchas human serum albumin, buffer substances such as phosphates, glycine,sorbic acid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

The pharmaceutical compositions of this invention may be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally, intradermally or via an implanted reservoir. Oraladministration or administration by injection is preferred. Thepharmaceutical compositions of this invention may contain anyconventional non-toxic pharmaceutically-acceptable carriers, adjuvantsor vehicles. The term parenteral as used herein includes subcutaneous,intracutaneous, intravenous, intramuscular, intra-articular,intrasynovial, intrasternal, intrathecal, intralesional and intracranialinjection or infusion techniques. Preferably, the route ofadministration is by injection, more preferably, the route ofadministration is intramuscular, intravenous or subcutaneous injectionand most preferably, the route of administration is intravenous orsubcutaneous injection.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween 80) and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluent,dispersant or similar alcohol.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, and aqueous suspensions and solutions. Inthe case of tablets for oral use, carriers which are commonly usedinclude lactose and corn starch. Lubricating agents, such as magnesiumstearate, are also typically added. For oral administration in a capsuleform, useful diluents include lactose and dried corn starch. Whenaqueous suspensions are administered orally, the active ingredient iscombined with emulsifying and suspending agents. If desired, certainsweetening and/or flavouring and/or colouring agents may be added.

The pharmaceutical compositions of this invention may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound of thisinvention with a suitable non-irritating excipient which is solid atroom temperature but liquid at the rectal temperature and therefore willmelt in the rectum to release the active components. Such materialsinclude, but are not limited to, cocoa butter, beeswax and polyethyleneglycols.

Topical administration of the pharmaceutical compositions of thisinvention is especially useful when the desired treatment involves areasor organs readily accessible by topical application. For applicationtopically to the skin, the pharmaceutical composition should beformulated with a suitable ointment containing the active componentssuspended or dissolved in a carrier. Carriers for topical administrationof the compounds of this invention include, but are not limited to,mineral oil, liquid petroleum, white petroleum, propylene glycol,polyoxyethylene polyoxypropylene compound, emulsifying wax and water.Alternatively, the pharmaceutical composition can be formulated with asuitable lotion or cream containing the active compound suspended ordissolved in a carrier. Suitable carriers include, but are not limitedto, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esterswax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Thepharmaceutical compositions of this invention may also be topicallyapplied to the lower intestinal tract by rectal suppository formulationor in a suitable enema formulation. Topically-transdermal patches arealso included in this invention.

The pharmaceutical compositions of this invention may be administered bynasal aerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

In one embodiment, the pharmaceutical composition may comprise aplurality of molecules of the invention, each molecule directed to exonskipping in a different exon. Alternatively, the pharmaceuticalcomposition may comprise a plurality of molecules of the invention, eachmolecule directed to exon skipping in the same exon.

In another embodiment, the pharmaceutical composition may comprise aplurality of vectors of the invention, each vector encoding a moleculedirected to exon skipping in a different exon. Alternatively, thepharmaceutical composition may comprise a plurality of vectors of theinvention, each vector encoding a molecule directed to exon skipping inthe same exon.

In yet another embodiment, the pharmaceutical composition may comprise amolecule and a vector, wherein the molecule and the molecule encoded bythe vector are directed to exon skipping in the same or different exons.

The present invention also provides a molecule of the invention for usein therapy.

Further, the present invention provides a molecule of the invention foruse in the amelioration of DMD.

The molecules of the present invention cause exon skipping in thedystrophin pre-mRNA. This causes a truncated but functional dystrophinprotein to be expressed which results in a syndrome similar to Beckermuscular dystrophy (BMD). Therefore, the symptoms of DMD will not becompletely treated but will be ameliorated so that they are potentiallyno longer life threatening.

The present invention also provides a method of ameliorating DMD in ahuman patient, the method comprising administering a therapeuticallyeffective amount of the molecule of the invention to the patient.

The particular molecule that is administered to the patient will dependon the location of the mutation or mutations present in the dystrophingene of the patient. The majority of patients have deletions of one ormore exons of the dystrophin gene. For example, if a patient is missingexon 44, the process of joining exon 43 to exon 45 will destroy theprotein, thus causing DMD. If exon 45 is skipped using a molecule of theinvention, the joining of exon 43 to exon 46 will restore the protein.Similarly, a patient with a deletion of exon 45 can be treated with amolecule to skip either exon 44 or exon 46. Further, a patient with adeletion of exons 45 to 52 inclusive (a large portion of the gene),would respond to skipping of exon 53.

In another aspect, the invention provides a kit for the amelioration ofDMD in a patient, the kit comprising a molecule of the invention andinstructions for its use. In one embodiment, the kit may contain aplurality of molecules for use in causing exon skipping in the same exonor a plurality of exons.

EXAMPLES Example 1

Here, the first detailed study of the role that AO target site variableshave on the efficacy of PMOs to induce skipping is reported. The resultsreported here should have an impact on the initial planning and designof AOs for future potential clinical trials.

Materials and Methods

Hybridization Analyses

Templates for the production of synthetic pre-mRNAs for exons 44, 45,46, 51, and 53 of the human dystrophin gene (DMD gene) were generated byPCR amplification from genomic clones of the exons, together withapproximately 500nt of upstream and downstream introns. PCR primersincorporated T7 RNA polymerase promoter sequences, such that pre-mRNAscould be produced by in vitro transcription. Pre-mRNAs were thensubjected to a hybridization screen against a spotted array of all 4096possible hexanucleotide sequences (Access Array 4000; Nyrion Ltd,Edinburgh UK). Binding of the pre-mRNA to specific spots on the arraywas detected by reverse transcriptase-mediated incorporation ofbiotinylated nucleotides by primer extension, followed by fluorescentlabelling. Scanning of the arrays followed by software analysis enabledsequences within the exons that were accessible to binding to thehexamer array to be identified. Using a hybridization assay, bindingaccessibility of each exons were analysed and hybridization peakidentified by AccessMapper software (Nyrion Ltd) (see FIG. 1 d).

AO Design

Overlapping AOs were designed to exons 44, 45, 46, 51, and 53 of thehuman DMD gene using the following information: putative SR proteinbinding domains as predicted by ESEfinder [20, 21], Rescue ESE [24] andPESX [22, 23] analyses of exon sequence; sequences accessible to bindingas determined by hybridization analyses (Nyrion); previously publishedwork [18, 19].

All AOs were synthesized as phosphorodiamidate morpholino oligos (PMOs)by Gene Tools LLC (Philomath Oreg., USA). To facilitate transfection ofthese uncharged oligonucleotides into cultured cells, the PMOs werehybridized to phosphorothioate-capped oligodeoxynucleotide leashes, asdescribed by Gebski et al., [12], and stored at 4° C.

The sequences of some of these PMOs were as follows:

(SEQ ID NO: 13) H44A30/1 - TGA AAA CGC CGC CAT TTC TCA ACA GAT CTG;(SEQ ID NO: 14) H44A30/2 - CAT AAT GAA AAC GCC GCC ATT TCT CAA CAG;(SEQ ID NO: 15) H44AB30/2 - TGT TCA GCT TCT GTT AGC CAC TGA TTA AAT;(SEQ ID NO: 16) H45A30/2 - CAG TTT GCC GCT GCC CAA TGC CAT CCT GGA;(SEQ ID NO: 17) H45A30/1 - TTG CCG CTG CCC AAT GCC ATC CTG GAG TTC;(SEQ ID NO: 18) H46A30/2 - TGC TGC TCT TTT CCA GGT TCA AGT GGG ATA;(SEQ ID NO: 19) H46A30/4 - CTT TTA GTT GCT GCT CTT TTC CAG GTT CAA;(SEQ ID NO: 20) H46A30/5 - CTT TTC TTT TAG TTG CTG CTC TTT TCC AGG;(SEQ ID NO: 21) H46A30/3 - TTA GTT GCT GCT CTT TTC CAG GTT CAA GTG;(SEQ ID NO: 22) H53A30/2 - CTG TTG CCT CCG GTT CTG AAG GTG TTC TTG;(SEQ ID NO: 23) H53A30/3 - CAA CTG TTG CCT CCG GTT CTG AAG GTG TTC;(SEQ ID NO: 24) H53A30/1 - TTG CCT CCG GTT CTG AAG GTG TTC TTG TAC.

Cell Culture and AO Transfection

Normal human primary skeletal muscle cells (TCS Cellworks, Buckingham,UK) were seeded in 6-well plates coated with 0.1 mg/ml ECM Gel(Sigma-Aldrich, Poole, UK), and grown in supplemented muscle cell growthmedium (Promocell, Heidelberg, Germany). Cultures were switched tosupplemented muscle cell differentiation medium (Promocell) whenmyoblasts fused to form visible myotubes (elongated cells containingmultiple nuclei and myofibrils). Transfection of PMOs was then performedusing the transfection reagent Lipofectin (Invitrogen, Paisley, UK) at aratio of 4 μl of Lipofectin per pg of PMO (with a range of PMOconcentrations tested from 50 to 500 nM, equivalent to approximately 0.5to 5 μg) for 4 hrs, according to the manufacturer's instructions. Alltransfections were performed in triplicate in at least two differentexperiments.

RNA Isolation and Reverse Transcriptase-Polymerase Chain ReactionAnalysis

Typically 24 h after transfection, RNA was extracted from the cellsusing the QIAshredder/RNeasy system (Qiagen, Crawley, UK) and ˜200 ngRNA subjected to RT-PCR with DMD exon-specific primers using theGeneScript kit (Genesys, Camberley, UK). From this 20 cycle reaction, analiquot was used as a template for a second nested PCR consisting of 25cycles. PCR products were analysed on 1.5% agarose gels inTris-borate/EDTA buffer. Skipping efficiencies were determined byquantification of the PCR products by densitometry using GeneToolssoftware (Syngene, Cambridge, UK).

Statistical Analysis

The non-parametric Mann-Whitney rank sum test was used to identifywhether parameters for effective PMOs were significantly different tothose for ineffective PMOs. Where data was calculated to fit a normaldistribution, the more powerful two-tailed Student's t-test wasperformed instead. Correlations were generated using the Spearmanrank-order test. To determine the strength of the combined significantparameters/design tools to design effective PMOs, linear discriminantanalysis was used [34], with the Ida function from the MASS package,using “effective” or “ineffective” as the two prior probabilities. TheIda function produces posterior probabilities for the two classes(effective and ineffective) for each PMO by leave-one-outclassification.

Results

PMO Design and Analysis of Bioactivity

A unique set of 66 PMOs has been designed to target exons 44, 45, 46,51, and 53 of the human gene for dystrophin. The design process for exon53 is depicted in FIG. 1, and has also been performed for the other fourexons (data not shown). The exon sequence was analysed for the presenceof exonic splicing enhancers (ESE) and exonic splicing suppressors orsilencers (ESS) and the outputs aligned for the three availablealgorithms, ESEfinder (FIG. 1 a) [20, 21], PESX (FIG. 1 b) [22, 23], andRescue ESE (FIG. 1 c) [24]. Hybridization array analysis was alsoperformed for each exon in vitro, as described in Materials and Methods.The peaks shown in FIG. 1 d indicate areas of the exon that are in aconformation able to hybridize to the array, and which may consequentlyprove more accessible to antisense AOs. The coincidence of ESEs, aspredicted by two or more algorithms, and hybridization peaks determinedexperimentally, was used to design arrays of 25 mer and, subsequently,30 mer PMOs, the positions of which are shown in FIG. 1 f. The bindingsites for 2′OMePS AOs described previously [18] are shown for comparison(FIG. 1 e).

Each PMO was tested in primary cultures of human skeletal muscle, intriplicate, in at least two experiments, and over a range ofconcentrations from 50 nM to 500 nM. Their bioactivity was determined byRT-PCR analysis, which showed a wide variation in the level of exonskipping induced (FIG. 2, and data not shown), ranging from 0% for h53C1(FIG. 1 f and FIG. 2, lane 2) to 80% for h53A30/3 (FIG. 1 f and FIG. 2,lane 6). Sequencing of the PCR products verified accurate skipping ofthe targeted exon (data not shown). The activity of each PMO at thestated optimal concentration is summarized in Table 1. Bioactivity isexpressed as a percentage of the skipped amplicon relative to total PCRproduct, as assessed by densitometry. Specific, consistent and sustainedexon skipping was evident for 44 of the 66 PMOs tested.

In Silico Analysis of PMOs

We then performed a retrospective in silico analysis of thecharacteristics of all 66 PMOs tested in this study, with respect to PMOlength, the distance of the PMO target site from the splice donor andacceptor sites, PMO-to-target binding energy and PMO-to-PMO bindingenergy, as calculated using RNAstructure2.2 software for the equivalentRNA-RNA interaction, and percentage GC content of the PMO, the resultsof which are summarized in Table 1. Also shown in Table 1 is thepercentage overlap of each PMO target site with sequences shown to beaccessible to binding, as determined experimentally by the hexamerhybridization array analysis. The relationship of PMO target site andRNA secondary structure was also examined using the program MFOLD [25](FIG. 3 and data not shown), with the percentage overlap of PMO targetsite with sequence predicted to be in open conformation by MFOLDanalysis given in Table 1. ESEfinder and SSF (http://www.umd.be/SSF/)software analysis of exon sequences revealed the positions of putativeSR protein binding motifs (SF2/ASF (by two algorithms), SC35, SRp40,SRp55, Tra2β and 9G8). The highest score over threshold for each SRprotein is given for each PMO in the columns on the right of Table 1.Also shown is the degree of overlap of each PMO target site with the ESEand ESS regions predicted by Rescue ESE and PESX.

Statistical Analysis of Design Parameters in Relation to PMO Bioactivity

For this statistical analysis, bioactive PMOs are considered to be thosewhich produce over 5% skipping, while those that produce less than 5%skipping are considered inactive. For each of the parameters listed inTable 1, comparison was made between bioactive and inactive PMOs usingthe non-parametric Mann-Whitney rank sum test, or, when it wasstatistically valid to do so, the parametric Student's t-test(two-tailed). The significant parameters are listed in Table 2.Considering the data as a whole, the variable which showed the highestsignificance to PMO bioactivity was the binding energy of the PMO to theexon (p=0.001); the most bioactive exons are predicted to bind better totheir target sites. Those PMOs that overlap with peaks identified by theexperimental hybridization array analysis are not significantly moreactive than those that do not (p=0.056), but when only the strongestpeak for each exon is considered, this parameter becomes highlysignificant (p=0.003). Distance of the PMO target site to the spliceacceptor site of the exon was also highly significant (p=0.004), withPMOs whose target site were closer to the acceptor site being moreactive. PMOs whose target sites showed coincidence with binding motifsfor the SR protein SF2/ASF (as defined by the BRCA1 algorithm of Smithet al. [21]) produced significantly greater skipping (p=0.026). PMOlength is also a significant parameter (p=0.017), with longer PMOs beingmore effective at inducing skipping. Boxplots of the significantvariables identified here are shown in FIG. 5. None of the othervariables considered in this study were shown to have any significanceto AO bioactivity.

To ascertain which parameters/design tools are the most powerful, wealso used the Mann-Whitney rank sum test to compare the most active PMOs(i.e. those that induce greater than 75% skipping of the target exon) tothose that were inactive (i.e. those that produce less than 5%skipping). Boxplots of the significant variables for this comparison areshown in FIG. 4. There is strong significance of overlap of the PMOtarget site with the strongest hybridization peak for each exon(p=0.002); more of the most bioactive PMOs had their target sitescoincident with sequences accessible to binding than those that wereinactive. This is reinforced by the observation that the target sites ofPMOs that produced over 75% skipping significantly overlapped more RNAthat was in open conformation, relative to inactive PMOs (p=0.025).Stronger binding between the PMO and its target exon, PMO length, andproximity of the target to the acceptor site are also significantparameters when comparing the most and least effective reagents.

Spearman's rank order correlation was used to establish potentialrelationships between design parameters and skipping bioactivity usingthe entire set of PMOs. This shows a strong correlation between skippingbioactivity and PMO-target binding energy (r_(s)=−0.618, p=0),percentage open conformation (r_(s)=0.275, p=0.0259), PMO length(r_(s)=0.545, p=0), distance from the splice acceptor site(r_(s)=−0.421, p=0), percentage overlap with the strongest hybridizationpeak (r_(s)=0.46, p=0), and overlap with an ESS sequence, as predictedby PESX (r_(s)=0.261, p=0.0348).

Linear Discriminant Analysis

This analysis was performed on all possible combinations of length,overlap with the SF2/ASF (BRCA1) motif, percentage overlap with areas ofopen conformation, percentage overlap with hybridization peak andPMO-target binding energy, i.e. PMO parameters and design tools thatshowed significance or borderline significance. Using length, SF2/ASF(BRCA1) motif and hybridization peak data, nine of the inactive PMOswere classified as bioactive and four bioactive PMOs were classified asinactive (Table 3). These four misclassified PMOs were 25 mers to exon46, three of which have borderline bioactivity, i.e. produce just 10%skipping, while the fourth produces about 20% skipping. Taken overall,this equates to 80% of the PMOs being predicted correctly when assessedaccording to their length, SF2/ASF (BRCA1) overlap and hybridizationpeak overlap. This would suggest that these parameters have thepotential to be effective design tools, with four out of every five PMOsdesigned to have these three properties likely to be bioactive. In linewith this, there was a distinct trend for PMOs being correctly assignedas bioactive with increased skipping bioactivity (see Table 3). Indeed,the PMOs with greatest bioactivity were all 30 mers (10/10), bound totheir target with a high binding energy of below −43.0 kD (9/10),overlapped by over 50% with areas of open conformation (7/10),overlapped with SF2/ASF (BRCA1) peak (8/10), and overlapped with ahybridization peak (7/10).

Discussion

Clinical studies using AOs to skip exon 51 to correct DMD deletions areprogressing well [11; F. Muntoni, Principal Investigator of MDEXConsortium, personal communication]. However since the mutations thatcause DMD are so diverse, skipping of exon 51 would have the potentialto treat just 24.6% of DMD patients on the Leiden DMD database [26]. Itis therefore imperative that pre-clinical optimization of AO targetsequence and chemistry is continually studied and improved. This studyhas examined the significance of design parameters for PMO-inducedskipping of exons 44, 45, 46, 51, and 53, which would have the potentialto treat, respectively, 11.5%, 15.8%, 8.4%, 24.6% and 13.5% of DMDpatients in the Leiden database [26; A. Aartsma-Rus, personalcommunication].

Specific skipping was observed for the five DMD exons studied here, withtwo-thirds of the PMOs tested being bioactive. This proportion ofbioactive AOs within a cohort has been reported previously [18, 19], butwe have induced high-level (i.e. greater than 75%) skipping in four ofthe five exons tested, some of which are achievable at relatively lowdoses of oligomer. The exception is exon 51, published previously [4],achieving a maximal skipping of 26%. The work of Wilton et al [19]demonstrated that only exons 51 and 53 can be skipped with highefficiency (>30% by their definition), and that exons 44, 45 and 46 areless “skippable” (less than 30% skipping). Furthermore, Aartsma-Rus etal [18] showed oligomers capable of high-level skipping (greater than amere 25%) for only exons 44, 46 and 51.

We provide here direct evidence that AO bioactivity shows a significantassociation with accessibility of its target site to binding. This isthe first study to assess sequences practically within the pre-mRNA thatare accessible to binding and then use them as an aid to AO design. Thedata we show underline the value of the hybridization analysis indetermining what are likely to be the most bioactive oligomers (i.e.those that produce greater than 75% skipping). As an example, if we lookat the data for oligomers developed for exon 45 [18], we see that thereis only one moderately effective (5-25%) reagent for this otherwiseunskippable exon. This oligomer is the only one of the six tested thatoverlaps with the strongest peak in our hybridization analysis. Thepartial nature of this overlap, combined with the short length of theoligomer, is likely to contribute to its relative weakness compared tothe PMOs we have developed here. In general, the 2′OMePS AOs displayingthe highest bioactivity in the work of Aartsma-Rus et al. [18] andWilton et al. [19], show some degree of overlap with the hybridizationpeaks that we have defined here for exons 45, 46 and 53.

Ease of skipping of certain DMD exons has been seen elsewhere [18] andmay be related to other factors affecting splicing, including strengthof splice donor and acceptor sites and branchpoint, and the size ofupstream and downstream introns, which may affect the order in whichexons are spliced together. There is the potential of using a cocktailof AOs to induce greater skipping of the more difficult to skip exons[27, 28].

Accessibility of the AO to its target site depends directly on thesecondary structure of the pre-mRNA, which has a major role indetermining AO bioactivity in cells. A study in which the structurearound an AO target site was changed revealed that AOs were unable toinvade very stable stem-loop structures and their antisense activity wasinhibited, but generally showed good activity when impeded by littlelocal structure [29]. Overlap of PMO target sites with openconformations in the folded RNA showed a weak association with PMObioactivity, which was more obvious when only the stronger PMOs wereconsidered in the statistical analysis. It is also possible that thereis selective pressure for SR binding sites to be located preferentiallyon these open secondary structures. The presumption is that binding ofbioactive PMOs to their target sites sterically block the binding ofimportant factors involved in RNA processing, resulting in exonskipping.

One of the PMO parameters with high significance was length; 30 mer PMOswere far superior to their 25 mer counterparts. The influence of 2′OMePSAO length on bioactivity has been reported elsewhere [30] and such anobservation for PMO-induced skipping of exon 51 has been reportedpreviously by us [4]. The more persistent action of longer PMOs wouldhave important cost and dose implications in the choice of AO forclinical trials. Longer AOs are likely to sterically block more of theregions that interact with the splicing machinery, but in general terms,the energy of binding of the longer PMO to its target would beincreased, which we showed to be the most significant parameter in AOdesign. The strong significance of the binding energy of PMO-targetcomplexes (i.e., free energy of AO-target compared to free energy of thetarget) and PMO length to bioactivity suggests that PMO bioactivitydepends on stability of the PMO-target complex, and implies thatbioactive PMOs act by interference of target RNA folding. Computationalanalysis revealed that the thermodynamics of binding of active PMOs totheir target site had a dramatic effect on the secondary foldedstructure of the RNA (data not shown). It is likely that these changesin secondary structure would have a profound effect on the binding of SRproteins to the RNA, thereby disrupting splicing, and exon skippingwould ensue.

Overlap of a PMO target site with a binding site motif for the SRprotein SF2/ASF (BRCA1), as predicted by ESEfinder, showed a significantassociation to PMO bioactivity. This partly confirms the work ofAartsma-Rus et al. [18], who observed marginally significantly higherESEfinder values for SF2/ASF and SC35 motifs for effective AOs whencompared to inactive AOs. SC35 and SF2/ASF motifs are the two mostabundant proteins assessed by ESEfinder. The reason why we do not seeany significance of overlap with SC35 motif to PMO bioactivity may bedue to the difference in AO chemistry used, and the number of AOsassessed. However Aartsma-Rus et al. [18] did note that not everybioactive AO has a high value for any of the SR protein binding motifs,and some inactive AOs have high values. The apparent weakness andunreliability of SR protein binding motifs as design tools for AOs maybe a reflection of the lack of precision of the predictive softwareused. Overlap of PMO target site with exonic splicing silencers appearsto show a correlation with bioactivity in Spearman's rank order testanalysis. Such a correlation would be counter-intuitive and the truesignificance questionable. Again the strength of the predictive softwareused may be in doubt. It should be noted that the software programmesused predict SR binding motifs on the linear exon sequence. Theavailability of these predicted motifs to bind SR proteins, or forbinding PMOs to disrupt the binding of these proteins, is directlyrelated to the folding of the pre-mRNA. The discrepancy in the relativesignificance of secondary RNA structure and SR protein binding motifsmay be due to active PMOs disrupting SR protein binding, not stericallybut indirectly, by altering the secondary pre-mRNA structure. A veryrecent study has shown the importance of co-transcriptional pre-mRNAfolding in determining the accessibility of AO target sites and theireffective bioactivity, and showed a direct correlation between AObioactivity and potential interaction with pre-mRNA [31].

It has been previously reported that ESE sites located within 70nucleotides of a splice site are more active than ESE sites beyond thisdistance [32]. Our results partially support this; PMOs with theirtarget site closer to the splice acceptor site are significantly morebioactive. However distance of the PMO target site to the splice donorsite showed no statistical significance to bioactivity. This bias hasbeen previously reported for the analyses of 2′OMePS AOs [18, 19], andmay be related to the demonstration, by Patzel et al. [33], of theimportance of an unstructured 5′ end of RNA in the initiation ofhybridization of oligonucleotide binding. This would suggest thattargeting any significant parameters located in the 5′ part of an exonmay increase the probability of designing a bioactive AO.

In conclusion, our findings show that no single design tool is likely tobe sufficient in isolation to allow the design of a bioactive AO, andempirical analysis is still required. However this study has highlightedthe potential of using a combination of significant PMOparameters/design tools as a powerful aid in the design of bioactivePMOs.

Linear discriminant analysis revealed that using the parameters of PMOlength, overlap with SF2/ASF (BRCA1) motif and hexamer arrayhybridization data in combination would have an 80% chance of designinga bioactive PMO, which is an exciting and suprising finding, and shouldbe exploited in further studies.

TABLE 1 Table 1: Table summarizing the characteristics of PMOs usedExon-PMO PMO-PMO Ends in % overlap Targeted Optimal % binding bindingopen Distance from with PMO exon conc. Skip^(a) Length % GC energyenergy % open^(b) loops^(b) donor acceptor hybrid peak h53B1 53 500 0 2528 −22.1 −12.1 53.3 1 119 68 0 h53C1 53 500 0 25 48 −32.4 −9.8 46.7 2 79108 0 h53C2 53 500 0 25 56 −31.3 −12.7 33.3 1 72 115 0 h53C3 53 500 0 2560 −34.6 −13.7 26.7 1 60 127 0 h53D1 53 500 0 25 52 −34.1 −13.4 30 1 39148 0 h45A30/4 45 500 0 30 43 −35.2 −7.5 40 1 53 93 100 h45A30/6 45 5000 30 53 −42.4 −26.9 46.7 2 9 137 100 h46A10 46 500 0 25 40 −35.3 −1.723.3 1 63 60 0 h46A30/6 53 500 0 30 40 −42.1 −10.1 56.7 0 5 113 0 h53D246 500 0.1 25 48 −36.5 −14.5 40 2 30 157 0 h46A5 53 500 0.2 25 36 −33.9−7.9 53.3 0 10 113 0 h53A6 53 500 0.3 25 48 −35.3 −8.5 43.3 2 138 49 92h53B2 53 500 0.6 25 48 −30.1 −11.3 23.3 1 108 79 0 h46A11 46 500 0.6 2520 −24.5 −1.5 43.3 0 0 143 0 h46A30/8 46 500 1.5 30 30 −34.2 −1.8 46.7 00 136 0 h45A30/7 45 500 1.6 30 50 −46.1 −4.7 73.3 0 0 158 100 h45A30/845 500 1.6 30 40 −39.3 −13.7 53.3 1 76 70 100 h53A3 53 500 2 25 56 −36.7−13.7 36.7 0 147 40 0 h46A9 46 500 2.1 25 28 −31.5 −7.6 36.7 1 109 14 0h53B3 53 500 3 25 48 −34.5 −5.5 48 2 98 89 0 h53D3 53 500 3.7 25 36−34.3 −11.2 40 1 18 169 0 h44B30/8 44 500 4.6 30 37 −28.3 −23.5 40 1 3484 0 h44B30/4 44 50 5 30 43 −38.2 −14.6 40 0 54 64 0 h46A6 46 100 5.4 2536 −31.5 −8 46.7 1 0 123 0 h46A8 46 500 5.4 25 32 −28.6 0 20 1 76 47 0h45A30/3 45 500 6.3 30 40 −35.5 −11.8 60 1 108 38 100 h53D5 53 500 7.925 36 −31.5 −3.3 66.7 1 0 187 0 h46A1 46 100 8.3 25 48 −35.7 −11.9 53.31 38 85 100 h53A5 53 250 9 25 48 −35.5 −8.5 43.3 2 141 46 100 h46A7 46500 9.1 25 32 −34.8 −5.6 36.7 1 123 0 0 h53A30/5 53 100 9.4 30 47 −42.4−11.3 46.7 1 141 41 100 h53A2 53 100 9.7 25 56 −36.1 −17.4 46.7 1 150 37100 h53A4 53 500 10.5 25 48 −34.3 −8.5 20 0 144 43 100 h45A30/5 45 50011.2 30 63 −44 −21.1 26.7 0 17 129 100 h53D4 53 500 12.3 25 32 −30.9−9.2 63.3 1 6 181 0 h53A1 53 100 12.7 25 52 −38.6 −17.4 50 2 153 34 92A25 51 250 14.9 25 36 −29.3 −11.6 66.7 2 146 62 0 h46A2 46 500 15.6 2544 −31.2 −10.6 56.7 1 33 90 100 h46A30/7 46 500 18.5 30 30 −34.2 −6.253.3 1 0 141 0 h46A4 46 100 21.2 25 44 −39.9 −6.3 56.7 2 20 103 46h44C30/2 44 50 22 30 33 −38 −7.4 36.7 1 7 111 0 h44B30/7 44 100 26 30 37−33.9 −10.9 26.7 1 39 79 0 h51A 51 500 26.3 30 43 −40.3 −15 70 1 137 650 h44B30/6 44 500 32.5 30 37 −34.6 −9.6 30 2 44 74 0 h44C30/3 44 500 3530 33 −38.9 −13.8 30 1 2 116 0 h44B30/1 44 100 35 30 33 −35.2 −7.1 66.71 69 49 0 h53A30/6 53 500 35.9 30 47 −42.3 −8.5 56.7 1 138 44 100h53A30/4 53 100 38.6 30 50 −43.4 −17.4 43.3 1 144 38 100 h44C30/1 44 10042 30 37 −41.1 −10.4 50 1 12 106 0 h46A3 46 100 49.7 25 48 −43.1 −5.256.7 2 28 95 100 h44A30/3 44 250 52.1 30 37 −42.5 −8.6 56.7 1 99 19 0h53A30/1 53 100 52.4 30 50 −48.1 −17.4 56.7 1 153 29 92 h44B30/3 44 50061 30 43 −35.4 −11.4 30 0 59 59 0 h44B30/5 44 500 63.3 30 40 −35.9 −14.630 1 49 69 0 h45A30/1 45 500 64.5 30 60 −49.7 −11 36.7 1 146 0 100h46A30/3 46 500 74.6 30 43 −49.8 −6.3 73.3 2 23 95 100 h46A30/1 46 50075.6 30 47 −43.5 −12.3 63.3 0 33 85 100 h46A30/5 46 500 76.7 30 40 −49.2−6.5 70 1 15 103 46 h53A30/3 53 100 80.1 30 53 −44.6 −17.4 53.3 1 147 35100 h44B30/2 44 500 80.5 30 37 −36.9 −10.7 50 1 64 54 0 h53A30/2 53 10087.2 30 53 −45.1 −17.4 63.3 1 150 32 100 h46A30/4 46 500 87.3 30 40−47.5 −6.3 73.3 2 20 98 85 h46A30/2 46 500 87.9 30 47 −49.1 −13.4 63.3 228 90 100 h45A30/2 45 500 91.4 30 60 −46.6 −13 20 1 142 4 100 h44A30/244 500 95 30 43 −44 −8.6 40 0 104 14 0 h44A30/1 44 250 97 30 47 −47.5−11.2 46.7 1 109 9 0 % overlap % overlap # Rescue with with ESE findervalues over threshold^(c) PMO ESE sites Rescue ESE PESE PESS SF2/ASFBRCA1 SC35 SRp40 SRp55 Tra2B 9G8 h53B1 5 56 40 40 0 9.26 3.62 10.66 05.06 1.1 h53C1 6 52 72 0 4.19 6.72 0 2.04 0 24.04 28.68 h53C2 1 24 60 04.19 6.72 10.2 4.38 0 0 8.28 h53C3 1 24 32 0 3.49 6.41 10.2 4.39 6.86 014.18 h53D1 4 40 32 0 0.52 0 18.68 0 6.86 0 12.71 h45A30/4 4 40 0 0 6.294.8 5.9 17.91 0 18.18 8.14 h45A30/6 4 40 0 0 11.64 7.34 5.04 1.38 0 7.2516.53 h46A10 7 60 48 8 2.21 0 2.7 2.88 0 5.11 23.85 h46A30/6 7 40 50 0 00 0 5.09 0 24.04 6.94 h53D2 6 44 32 0 0.52 1.8 18.68 0.42 0 0 12.71h46A5 7 48 44 0 0 0 0 5.09 0 24.04 6.94 h53A6 2 36 28 32 6.58 7.26 0 0 07.25 11.9 h53B2 5 60 60 0 0 9.26 3.62 4.73 0 5.06 8.28 h46A11 2 36 12 520 0 0 1.02 0 0 2.04 h46A30/8 1 27 27 43 0 0 0 1.02 0 0 2.04 h45A30/7 947 0 0 6.34 7.34 0 0.6 0 18.18 8.14 h45A30/8 4 47 0 0 0 0 5.9 2.4 018.18 17.14 h53A3 3 32 60 0 6.58 7.26 0 3.12 0 7.25 11.9 h46A9 8 48 25 00 7.87 0 0 0 24.04 7.14 h53B3 8 72 64 0 3.49 9.26 3.44 4.73 0 24.0428.68 h53D3 9 64 0 0 0 1.8 0 6.95 0 24.04 10.49 h44B30/8 7 57 27 13 2.858.64 7.06 1.38 0 10.92 19.02 h44B30/4 8 47 37 27 1.98 8.64 6.14 10.12 07.25 8.28 h46A6 7 72 64 0 0 0 0 5.09 0 24.04 6.94 h46A8 5 56 24 60 2.210 3.56 2.88 0 0 23.68 h45A30/3 9 87 30 0 0 6.18 3.07 4.73 0.45 24.0428.68 h53D5 14 92 44 0 8.5 11.95 0 7.67 0.33 24.04 7.14 h46A1 3 20 40 02.62 20.26 6.63 6.17 0 0 5.12 h53A5 3 36 36 20 6.58 7.26 0 3.12 0 7.2511.9 h46A7 9 64 44 0 0 0 6.02 4.2 0 24.04 28.68 h53A30/5 5 47 47 17 6.587.26 0 3.12 0 7.25 11.9 h53A2 4 32 72 0 6.58 7.26 0 3.12 0 7.25 19.02h53A4 4 28 48 8 6.58 7.26 0 3.12 0 7.25 11.9 h45A30/5 2 23 0 0 11.6413.49 5.04 1.38 0 7.25 16.53 h53D4 16 96 24 0 8.5 11.95 0 7.67 0.3324.04 7.14 h53A1 7 56 84 0 6.58 7.26 0 3.12 0 24.04 19.02 A25 1 24 12 321.22 13.72 0 0 0 0 0 h46A2 5 40 40 0 2.62 20.26 6.63 6.17 0 13.11 5.12h46A30/7 2 20 10 43 0 0 0 1.02 0 0 2.1 h46A4 8 60 40 0 0 0 0 5.09 024.04 6.94 h44C30/2 3 33 10 63 0.52 5.72 0 0 0 9.46 5.6 h44B30/7 6 40 3027 2.85 8.64 7.06 1.38 0 10.92 19.02 h51A 2 40 3 27 1.22 13.72 0 0 0 04.45 h44B30/6 8 37 20 27 2.85 8.64 0 1.92 0 10.92 19.02 h44C30/3 2 33 063 0 0 0 6.44 0 9.46 5.6 h44B30/1 6 67 33 30 0 0 6.14 10.12 0 10.92 8.28h53A30/6 5 48 37 27 6.58 7.26 0 3.12 0 7.25 11.9 h53A30/4 4 43 57 7 6.587.26 0 3.12 0 7.25 11.9 h44C30/1 3 43 27 63 0.52 5.72 7.06 0 0 9.46 5.6h46A3 5 40 40 0 2.62 20.26 6.63 6.17 0 13.11 5.12 h44A30/3 3 23 0 77 013.26 0 0 0 0 11.3 h53A30/1 9 60 86 0 6.58 7.26 0 3.12 0 24.04 19.02h44B30/3 5 47 37 33 0 0 6.14 10.12 0 7.25 8.28 h44B30/5 10 63 37 27 1.988.64 6.14 1.92 0 10.92 19.02 h45A30/1 2 0 0 6.7 3.43 8.64 5.16 3.54 3.570 20.56 h46A30/3 5 40 33 0 0 0.57 0 6.17 0 13.11 5.12 h46A30/1 5 33 33 02.62 20.26 6.63 6.17 0 13.11 5.12 h46A30/5 12 67 50 0 0 0 0 5.09 0 24.046.94 h53A30/3 6 43 67 0 6.58 7.26 0 3.12 0 24.04 19.02 h44B30/2 5 50 3737 0 0 6.14 10.12 0 7.25 8.28 h53A30/2 8 53 77 0 6.58 7.26 0 3.12 024.04 19.02 h46A30/4 8 50 43 0 0 0.57 0 5.09 0 24.04 5.12 h46A30/2 5 3333 0 2.62 20.26 6.63 6.17 0 13.11 5.12 h45A30/2 0 0 0 20 3.43 10.41 5.163.54 3.57 0 20.56 h44A30/2 3 27 0 63 0 13.26 0 0 0 0 11.3 h44A30/1 4 430 47 0 13.26 0 2.76 0 0 11.3 PMOs are ranked in order of efficacy andcharacteristics of the PMOs and their target sites listed.^(a)calculated as % skipped amplicon relative to total amplicon (i.e.skipped plus full length) as assessed by densitometric analysis ofRT-PCR gels. ^(b)calculated as % on PMO target site in open structureson predicted RNA secondary structure obtained using MFOLD analysis. Theposition of the PMO target sites relative to open loops in the RNAsecondary structure is listed (0 = no ends in open loops, 1 = one end inan open loop, 2 = both ends in open loops). ^(c)In analyses, SR bindingsites were predicted using splice sequence finder(http://www.umd.be/SSF/) software. Values above threshold are given forPMOs whose target sites cover 50% or more of potential SR binding sitesfor SF2/ASF, BRCA1, SC35, SRp40, SRp55, Tra2β and 9G8.

TABLE 2 Table 2: The correlation of significant design parameters andPMO target site properties to skipping efficacy % overlap with % overlapwith PMO-target % open Distance from hybridisation strongest hybrid. %overlap with Comparison binding energy conformation Length acceptor sitepeak peak BRCA1 motif Ineffective vs 0.001 0.094 0.017 0.004 0.056 0.0030.026 Effective Ineffective vs 0.534 0.288 1 0.163 0.107 0.034 0.2055-25% skip Ineffective vs 0.02 0.316 0.014 0.067 0.614 0.195 0.07925-50% skip Ineffective vs 0.002 0.438 0.012 0.005 0.352 0.084 0.34150-75% skip Ineffective vs <0.001 0.025 0.002 0.003 0.045 0.002 0.09175-100% skip Ineffective vs >50% <0.001 0.052 <0.001 <0.001 0.05 0.0050.046 skip Spearmans correlation −0.618 0.275 0.545 −0.421 0.258 0.460.261 coefficient Spearmans p value 0 0.0259 0 0 0.0363 0 0.0341 Toestablish the significance of design parameters and PMO target siteproperties to bioactivity, Mann-Whitney rank sum test analysis wasperformed for each, comparing ineffective (inactive) PMOs to thedifferent groups of PMOs, subdivided (in the column headed “Comparison”)according to bioactivity (efficacy). Criteria with p-values less than0.05 in one or more comparisons are shown. The correlation of thesevariables to bioactivity is confirmed by Spearman rank order testanalysis, for which Spearman correlation coefficients and p-values aregiven.

TABLE 3 Table 3: Linear discriminant analysis of effective andineffective PMOs Classification Average Group Effective IneffectiveTotal Error rate score Effective 40 4 44 0.09 0.741 Ineffective 9 13 220.41 0.512   0-5% skip 9 13 22 0.41 0.512  5-25% skip 16 4 20 0.2 0.621 25-50% skip 9 0 9 0 0.806  50-75% skip 6 0 6 0 0.827 75-100% skip 10 010 0 0.857 Linear discriminant analysis [34] was used to predict theclassification of PMOs on the basis of their PMO-target binding energy,overlap of PMO target site with a hybridization peak, and overlap of PMOtarget site with an ASF/SF2 (BRCA1) motif. PMOs have been grouped on thebasis of their experimental bioactivity (“Group” column), and PMOswithin each group predicted as “Effective” (bioactive) or “Ineffective”(inactive), as indicated by the column headings, according to theparameters used in the statistical analysis. The error rate for wronglyclassifying aPMO, and the average score are given for each subgroup ofPMO.

Example 2

Here, the inventors show the comparative analysis of a series of PMOstargeted to exon 53, skipping of which would have the potential to treata further 8% of DMD patients with genomic deletions on the Leidendatabase compared to skipping of exon 51 which has the potential totreat 13% of DMD patients [37]. An array of overlapping PMOs weredesigned for the targeting of exon 53 as described previously [38].These were all tested initially in normal human skeletal muscle cells(hSkMCs), since these are more readily available than patient cells.PMOs that showed greatest skipping efficacy were further tested in cellsfrom a DMD patient with a relevant deletion (del 45-52). The PMOs withgreatest efficacy, in terms of concentration and stability, wereevaluated by performing dose-response and time-course studies. Findingsfrom these experiments were supported by in vivo studies in a mousemodel transgenic for the entire human dystrophin locus [8].Collectively, this work suggests that one particular PMO (A, h53A30/1,+30+59) produced the most robust skipping of exon 53, and should beconsidered the sequence of choice for any upcoming PMO clinical trial.

Materials and Methods

AO Design

Twenty-three overlapping AOs to exon 53 were designed as described abovein Example 1.

Cell Culture and AO Transfection

Transfections were performed in two centres (Royal Holloway, London UK(RHUL) and UCL Institute of Child Health, London UK (UCL)) and by twodifferent methods (liposome-carrier of leashed PMOs in normal cells(RHUL), and by nucleofection of naked PMOs in patient cells (UCL)). AOswere transfected into normal human primary muscle cells (TCS Cellworks,Buckingham, UK) and into patient primary skeletal muscle culturesobtained from muscle biopsies taken at the Dubowitz Neuromuscular Unit,UCL Institute of Child Health (London, UK), with the approval of theinstitutional ethics committee. Normal hSkMCs were cultured andtransfected with leashed PMOs, using 1:4 lipofectin, as describedpreviously [4]. To minimize any influence of leash design on PMO uptakeand subsequent bioactivity, the DNA sequences in the leashes were of thesame length (17 mers for the 25 mer PMOs or 20 mers for the 30 mer PMOs)and were completely complementary to the 3′-most 17 or 25 nt of eachPMO. The phosphorothioate caps of 5 nt at each end were notcomplementary to the PMOs, and had the same sequences for every leash.

DMD Patient Primary Myoblast Culture

Skeletal muscle biopsy samples were taken from a diagnostic biopsy ofthe quadriceps from a DMD patient with a deletion of exons 45-52.Informed consent was obtained before any processing of samples. Muscleprecursor cells were prepared from the biopsy sample by sharp dissectioninto 1 mm³ pieces and disaggregated in solution containing HEPES (7.2mg/ml), NaCl (7.6 mg/ml), KCl (0.224 mg/ml) Glucose (2 mg/ml) Phenol Red(1.1 ng/ml) 0.05% Trypsin-0.02% EDTA (Invitrogen, Paisley, UK) indistilled water, three times at 37° C. for 15 minutes in Wheaton flaskswith vigorous stirring. Isolated cells were plated in non-coated plasticflasks and cultured in Skeletal Muscle Growth Media (Promocell,Heidelberg, Germany) supplemented with 10% Foetal Bovine Serum (PAALaboratories, Yeovil, UK), 4 mM L-glutamine and 5 μg/ml gentamycin(Sigma-Aldrich, Poole, UK) at 37° C. in 5% CO₂.

Nucleofection of DMD Primary Myoblasts

Between 2×10⁵ and 1×10⁶ cells/ml were pelleted and resuspended in 100 μlof solution V (Amaxa Biosystems, Cologne, Germany). The appropriate PMOto skip exon 53 was added to the cuvette provided, sufficient to givethe concentrations described, followed by the cell suspension, andnucleofected using the Amaxa nucleofector 2, program B32. 500 μl ofmedia was added to the cuvette immediately following nucleofection. Thissuspension was transferred to a 6 well plate in differentiation medium.Nucleofected cells were maintained in differentiation media for 3-21days post treatment before extraction of RNA or protein.

Lactate Dehydrogenase Cytotoxicity Assay

A sample of medium was taken 24 hours post-transfection to assesscytotoxicity by release of lactate dehydrogenase (LDH) into the medium,using the LDH Cytotoxicity Detection Kit (Roche, Burgess Hill, UK),following the manufacturer's instructions. The mean of three readingsfor each sample was recorded, with medium only, untreated and deadcontrols. The readings were normalised for background (minus mediumonly) and percentage toxicity expressed as[(sample-untreated)/(dead-untreated)×100].

RNA Isolation and Reverse Transcription Polymerase Chain ReactionAnalysis

As with cell culture, two different techniques were used in the twocentres involved in this study for isolating RNA and its analysis byRT-PCR, as described previously [4]. PCR products were analysed on 1.5%(w/v) agarose gels in Tris-borate/EDTA buffer. Skipping efficiencieswere determined by quantification of the full length and skipped PCRproducts by densitometry using GeneTools software (Syngene, Cambridge,UK).

Sequence Analysis

RT-PCR products were excised from agarose gels and extracted with aQIAquick gel extraction kit (Qiagen, Crawley, UK). Direct DNA sequencingwas carried out by the MRC Genomics Core Facility.

Western Blot Analysis of Dystrophin Protein

DMD patient cells, transfected as described and cultured indifferentiation medium, were harvested 7, 14 or 21 dayspost-transfection. 4×10⁵ cells were pelleted and resuspended in 50 μl ofloading buffer (75 mM Tris-HCl pH 6.8, 15% sodium dodecyl sulphate, 5%β-mercaptoethanol, 2% glycerol, 0.5% bromophenol blue and complete miniprotease inhibitor tablet). Samples were incubated at 95° C. for 5minutes and centrifuged at 18,000×g for 5 minutes. 20 μl of sample wasloaded per well in a 6% polyacrylamide gel with 4% stacking gel. Proteinfrom CHQSB cells differentiated for 7 days was used as a positivecontrol for dystrophin. Gels were electrophoresed for 5 hours at 100Vbefore blotting on nitrocellulose membrane at 200 mA overnight on ice.Blots were stained with Protogold to assess protein loading, thenblocked in 10% non-fat milk in PBS with 2% tween (PBST) for 3 hours.Blots were probed with antibodies to dystrophin, NCL-DYS1 (Vector Labs,Peterborough, UK) diluted 1:40 and to dysferlin, Hamlet1 (Vector Labs)diluted 1:300 in 3% non-fat milk/PBST. An anti-mouse, biotinylatedsecondary antibody (diluted 1:2000; GE Healthcare, Amersham, UK) andstreptavidin/horse radish peroxidise conjugated antibody (1:10,000;Dako, Ely, UK) allowed visualisation in a luminol-HRP chemiluminescencereaction (ECL-Plus; GE Healthcare) on Hyperfilm (GE Healthcare), exposedat intervals from 10 seconds to 4 minutes.

Transgenic Human DMD Mice

A transgenic mouse expressing a complete copy of the human DMD gene hasbeen generated [8, 39]. Experiments were performed at the LeidenUniversity Medical Center, with the authorization of the AnimalExperimental Commission (UDEC) of the Medical Faculty of LeidenUniversity as described previously [4].

Results

Twenty-three PMOs were designed to target exon 53, as describedpreviously [38]. Briefly, SR protein binding motifs, RNA secondarystructure and accessibility to binding as determined by hexamerhybridization array analysis, were used as aids to design (FIG. 1).Table 4 summarises the names and target sequence characteristics ofthese PMOs. These PMOs were initially characterized in normal humanskeletal muscle cells (at RHUL). The most active were then directlycompared to the PMO targeting the sequence previously identified as mostbioactive by Wilton et al. [19] in exon 53-skippable patient cells (atUCL), and in the humanised DMD mouse (at LUMC).

Comparison of PMOs to Exon 53 in Normal Human Skeletal Muscle Cells

An array of seventeen 25 mer leashed PMOs were transfected, at aconcentration of 500 nM, into normal human skeletal muscle myoblastcultures using lipofectin. Of these seventeen, only four producedconsistent levels of exon skipping considered to be above backgroundi.e. over 5% skipping [38], as assessed by densitometric analysis (FIG.6 a). These were PMO-A, -B, -C and -D, which targeted exon 53 atpositions +35+59, +38+62, +41+65 and +44+68 respectively. The levels ofexon skipping produced were as follows: PMO-A, 12.7%; PMO-B, 9.7%;PMO-C, 10.5%; and PMO-D, 9.0%. When nucleofection was used as a means ofintroducing naked PMOs into the cells, higher levels of exon skippingwere observed for PMO-A and PMO-B only, with 300 nM doses producing41.2% and 34.3% exon skipping, respectively. The superiority ofnucleofection over lipofection has been observed by others (Wells etal., in preparation). However no exon skipping was evident followingnucleofection with any of the other naked 25 mer PMOs tested (data notshown).

A 3 nt-stepped array of 30 mer PMOs was then designed to target theregion of exon 53 (position +30 to +74) associated with exon skippingactivity by the 25 mer PMOs. Following lipofection into normal humanskeletal muscle myoblast cultures at a concentration of 500 nM, PMO-G(+30+59), PMO-H (+33+62), PMO-I (+36+65), PMO-J (+39+68) and PMO-K(+42+71) gave reproducible exon skipping above background (FIG. 6 b),while PMO-L (+45+74) was inactive. The levels of exon skipping producedwere as follows: PMO-G, 37.1%; PMO-H, 44.5%; PMO-I, 27.4%; PMO-J, 33.0%;and PMO-K, 13.0%. The concentration dependence of exon skipping by themore active 30 mer PMOs was examined further (FIG. 7 a). PMO-H and PMO-Iwere able to produce convincing skipping at concentrations as low as 25nM, while PMO-G was active at 50 nM and PMO-J at 75 nM. The exonskipping produced by these 30 mer PMOs was shown to be persistent,surviving the lifetime of the cultures (14 days) (FIG. 7 b and data notshown). When unleashed 30 mer PMOs were introduced into normal musclecultures by nucleofection, high levels of exon skipping were alsoobserved. For example, at 300 nM, PMO-G and PMO-H gave over 80% skippingof exon 53 (data not shown).

Comparison of PMOs to Exon 53 in DMD Patient Cells

The PMOs, both 25 mer and 30 mer, that produced the highest levels ofDMD exon 53 skipping in normal skeletal muscle cultures, were thencompared to each other for bioactivity in DMD patient (del 45-52) cells,and were also compared to an additional reagent, PMO-M (+39+69),described previously [19]. This comparative evaluation was performed ina blinded fashion. When tested and compared directly at 300 nM doses bynucleofection, PMO-G, PMO-H and PMO-A were most active producing in theorder of 60% exon skipping (FIG. 8). The other PMOs tested produced thefollowing exon skipping levels: PMO-I, 45%; PMO-B, 41%; PMO-J, 27%;PMO-M, 26%. All the other PMOs tested gave exon skipping at lower levelsof between 10 and 20%.

When the concentration dependence of exon skipping was examined for themost bioactive PMOs, levels approaching 30% were evident for PMO-G andPMO-H at concentrations as low as 25 nM (FIG. 9 a, b). Similar levels ofskipping were only achieved by PMO-A, PMO-B and PMO-M at 100 nM, whilePMO-I needed to be present at 200 nM to produce over 30% exon skipping(FIG. 9 a, b). There was no evidence that any of the PMOs tested causedcellular cytotoxicity relative to mock-transfected controls, as assessedby lactate dehydrogenase release into culture medium (results notshown). The exon skipping produced by the six most bioactive PMOs wasshown to be persistent, lasting for up to 10 days after transfection,with over 60% exon skipping observed for the lifetime of the culturesfor PMO-A, PMO-G and PMO-H (FIG. 10 a, b). Exon skipping was shown topersist for 21 days for PMO-A and PMO-G (FIG. 10 c).

Western blot analysis of DMD patient (dcl 45-52) cell lysates, treatedin culture with the most bioactive 25 mers (PMO-A and PMO-B) and longerPMOs (PMO-G, PMO-H, PMO-I and PMO-M) is shown in FIG. 10 e. De novoexpression of dystrophin protein was evident with all six PMOs, but wasmost pronounced with PMO-H, PMO-I, PMO-G and PMO-A, producing 50%, 45%,33% and 26% dystrophin expression, respectively, relative to thepositive control, and seemingly weakest with PMO-B and PMO-M (11% and17% dystrophin expression respectively, relative to the positivecontrol). However, the limitations of quantifying Western blots of thisnature should be taken into account when interpreting the data.

Comparison of PMOs to Exon 53 in Humanised DMD Mouse

The hDMD mouse is a valuable tool for studying the processing of thehuman DMD gene in vivo, and as such provides a model for studying the invivo action of PMOs, prior to clinical testing in patients. PMO-A,PMO-G, PMO-H, PMO-I and PMO-M were injected into the gastrocnemiusmuscle of hDMD mice, and RNA extracted from the muscles was analysed forexon 53 skipping (FIG. 11). Skipping of exon 53 is evident for each ofthe PMOs tested; 8% for PMO-A, 7.6% for PMO-I, 7.2% for PMO-G, but to aslightly lower level of 4.8% for PMO-H. PMO-M produced exon skippinglevels of less than 1%, which is the detection threshold for the systemused.

It should be noted that the levels of exon skipping by each particularPMO was variable. This has been reported previously [8], and is likelyto be due to the poor uptake into the non-dystrophic muscle of the hDMDmouse. However this does not compromise the importance of the findingthat the PMOs tested here are able to elicit the targeted skipping ofexon 53 in vivo.

Of the 24 PMOs tested, six (PMO-A, PMO-B, PMO-G, PMO-H, PMO-I and PMO-M)produced over 50% targeted skipping of exon 53 either in normal myotubesor in patient myotubes or both. The characteristics of these active PMOsand their target sites are summarised in Table 4. They all showed strongoverlap (92%-100%) with the sequence shown to be accessible to bindingon the hybridization array analysis, had similar GC content (50%-56%),but varying degrees of overlap (32%-60%) with ESE sites as predicted byRescue ESE analysis, varying degrees of overlap with ESE sites and ESSsites (60%-86% and 0%-10%, respectively) as predicted by PESX analysis,and all showed overlap with two SR binding motifs (SF2/ASF, as definedby the

BRCA1 algorithm, and SRp40). It should be noted that PMO-J, -K, -L and-M had a common SNP of exon 53 (c7728C>T) in the last, fourth to last,seventh to last and second to last base, respectively of their targetsites. There is the potential that this allelic mismatch could influencethe binding and bioactivity of these PMOs. However, the more active PMOs(-A, -B, -G, -H and -I) all had their target sites away from the SNP,and the possible effect of a mismatch weakening binding and bioactivityis removed, and allows definitive comparisons between these PMOs to bemade.

Discussion

The putative use of AOs to skip the exons which flank out-of-framedeletions is fast becoming a reality in the experimental intervention ofDMD boys. Indeed the restoration of dystrophin expression in the TAmuscle of four patients, injected with a 2′OMePS AO optimised to targetexon 51 of the DMD gene, has been reported recently [11]. Moreover aclinical trial using a PMO targeting exon 51 has recently been completedin seven DMD boys in the UK (Muntoni et al, in preparation). However,the targeted skipping of exon 51 would have the potential to treat only13% of DMD patients with genomic deletions on the Leiden database [37].There is therefore a definite requirement for the optimisation of AOs totarget other exons commonly mutated in DMD.

Although there have been many large screens of AO bioactivity in vitro[18, 19, 38, 40], no definite rules to guide AO design have becomeapparent. Previous studies in the mdx mouse model of DMD showed that AOsthat targeted the donor splice site of exon 23 of the mouse DMD generestored dystrophin expression [7]. However the targeting of AOs to thedonor splice sites of exon 51 of the human DMD gene was ineffective atproducing skipping [4], and it has been suggested that the‘skippability’ of human DMD exons has no correlation with the predictedstrength of the donor splice site [41]. It has been reported that exonskipping could be induced by the targeting of AOs to exonic splicingenhancer (ESE) motifs [18, 40]. These motifs are recognised by SRproteins, which facilitate exon splicing by recruiting splicingeffectors (U1 and U2AF) to the donor splice site (reviewed by Cartegniet al.) [42]. However these motifs are divergent, poorly defined, theiridentification complex, and their strength as AO design tools dubious[38].

A comparative study of 66 PMOs designed to five different DMD exonsdemonstrated the significance of RNA secondary structure in relation toaccessibility of the PMO target site and subsequent PMO bioactivity[38], as assessed by mfold software prediction of secondary structure[25], and a hybridization screen against a hexamer array [38]. PMOs thatbound to their target more strongly, either as a result of being longeror in being able to access their target site more directly, weresignificantly more bioactive. The influence of AO length on bioactivityhas been reported elsewhere [4, 30], and is further confirmed in thepresent study; all 30 mers tested were more bioactive relative to their25 mer counterpart. The fact that 30 mer PMOs were more bioactive than25 mer PMOs targeted to the same open/accessible sites on the exon,would suggest that strength of binding of PMO to the target site may bethe most important factor in determining PMO bioactivity. Thesethermodynamic considerations have also been reported in a complementarystudy of 2′OMePS AOs [40]. However, it has also been reported that twooverlapping 30 mers were not as efficient as a 25 mer at skipping mouseexon 23, indicating that oligomer length may only be important in somecases [4].

To ensure that the analysis of PMOs for the targeted skipping of exon 53was not biased by any particular design strategy, seventeen 25 mer PMOswere designed to cover the whole of exon 53, with stepwise arrays oversuggested bioactive target sites, and then subsequently six 30 mer PMOswere designed to target the sequence of exon 53 that showed anassociation with exon skipping for the 25 mers tested. PMOs weredesigned and tested independently by two different groups (at RHUL andUWA), and then efficacy of the best thirteen sequences confirmed by twoother independent groups (at UCL and LUMC). Such a collaborativeapproach has been used previously as a way of validating targetsequences in DMD [4]. Human myoblasts allowed the controlled in vitrocomparison of PMO sequences, and confirmation of skipping of exon 53 atthe RNA level by certain PMOs in both normal cells and, perhaps moreimportantly, in DMD patient cells with a relevant mutation. Theseresults were further borne out by the expression of dystrophin proteinin the DMD cells treated with specific PMOs. Use of the humanised DMDmouse provided an in vivo setting to confirm correct exon exclusionprior to any planned clinical trial. The combined use of these threedifferent systems (normal cells, patient cells and hDMD mouse) as testsof PMO bioactivity provided a reliable and coherent determination ofoptimal sequence(s) for the targeted skipping of exon 53.

When considering the data presented here as a whole, the superiority ofthe PMO targeting the sequence +30+59 (PMO-G, or h53A30/1), is stronglyindicated. In normal myoblasts, nucleofection of PMO-G (300 nM) andliposomal-carrier mediated transfection of leashed PMO-G (500 nM)produced over 80% and over 50% skipping of exon 53, respectively,implying that it acts extremely efficiently within the cell. This wasconfirmed in patient cells. Indeed, this PMO generates the highestlevels of exon skipping in patient cells over a range of concentrations(up to 200 nM) and, most important therapeutically, exerts its activityat concentrations as low as 25 nM. The exon skipping activity of thisPMO is also persistent, with over 70% exon skipping for 7 days inculture, and over 60% exon skipping for up to three weeks. This wouldhave important safety and cost implications as a genetic therapy for DMDpatients with the appropriate deletions. PMO-G was also shown to skipexon 53 correctly in vivo. These RNA results were further confirmed bythe detection of dystrophin protein at a high level in protein extractsfrom patient cells treated with PMO-G. Previous studies by the Leidengroup [18] suggest that the optimal 2′OMePS AO is targeted to thesequence +46+63 of exon 53, producing exon skipping in up to 25% oftranscripts in cultured cells and 7% in the hDMD mouse. This 2′OMePS AOshows some degree of overlap with the optimal PMOs reported here whichstrengthens our findings. The reason that our optimal PMO is morespecific could be a (combined) consequence of the different AOchemistries, length of AO used, and the absolute target site of AO.

The sequence h53A30/1 we have identified appears to be more efficientthan any of the previously reported AOs designed to skip exon 53 of theDMD gene, and this PMO therefore represents, at the present time, theoptimal sequence for clinical trials in DMD boys.

TABLE 4 Table 4: Table summarizing the characteristics of PMOs used % %overlap overlap Exon-PMO PMO-PMO Ends in with # Rescue with Positionbinding binding % open hybrid. ESE Rescue PMO Start End % GC energyenergy open^(b) loops^(b) peak sites ESE A h53A1 +35 +59 52 −38.6 −17.450 2 92 7 56 B h53A2 +38 +62 56 −36.1 −17.4 46.7 1 100 4 32 C h53A3 +41+65 56 −36.7 −13.7 36.7 0 0 3 32 D h53A4 +44 +68 48 −34.3 −8.5 20 0 1004 28 E h53A5 +47 +71 48 −35.5 −8.5 43.3 2 100 3 36 F h53A6 +50 +74 48−35.3 −8.5 43.3 2 92 2 36 N h53B1 +69 +93 28 −22.1 −12.1 53.3 1 0 5 56 Oh53B2 +80 +104 48 −30.1 −11.3 23.3 1 0 5 60 P h53B3 +90 +114 48 −34.5−5.5 48 2 0 8 72 Q h53C1 +109 +133 48 −32.4 −9.8 46.7 2 0 6 52 R h53C2+116 +140 56 −31.3 −12.7 33.3 1 0 1 24 S h53C3 +128 +152 60 −34.6 −13.726.7 1 0 1 24 T h53D1 +149 +173 52 −34.1 −13.4 30 1 0 4 40 U h53D2 +158+182 48 −36.5 −14.5 40 2 0 6 44 V h53D3 +170 +194 36 −34.3 −11.2 40 1 09 64 W h53D4 +182 +206 32 −30.9 −9.2 63.3 1 0 16 96 X h53D5 +188 +212 36−31.5 −3.3 66.7 1 0 14 92 G h53A30/1 +30 +59 50 −48.1 −17.4 56.7 1 92 960 H h53A30/2 +33 +62 53 −45.1 −17.4 63.3 1 100 8 53 I h53A30/3 +36 +6553 −44.6 −17.4 53.3 1 100 6 43 J h53A30/4 +39 +68 50 −43.4 −17.4 43.3 1100 4 43 K h53A30/5 +42 +71 47 −42.4 −11.3 46.7 1 100 5 47 L h53A30/6+45 +74 47 −42.3 −8.5 56.7 1 100 5 48 M H53A +39 +69 52 −48.5 −17.4 48.42 100 4 45 % overlap with ESE finder values over threshold^(c) PESE PESSSF2/ASF BRCA1 SC35 SRp40 SRp55 Tra2B 9G8 A 84 0 6.58 7.26 0 3.12 0 24.0419.02 B 72 0 6.58 7.26 0 3.12 0 7.25 19.02 C 60 0 6.58 7.26 0 3.12 07.25 11.9 D 48 8 6.58 7.26 0 3.12 0 7.25 11.9 E 36 20 6.58 7.26 0 3.12 07.25 11.9 F 28 32 6.58 7.26 0 0 0 7.25 11.9 N 40 40 0 9.26 3.62 10.66 05.06 1.1 O 60 0 0 9.26 3.62 4.73 0 5.06 8.28 P 64 0 3.49 9.26 3.44 4.730 24.04 28.68 Q 72 0 4.19 6.72 0 2.04 0 24.04 28.68 R 60 0 4.19 6.7210.2 4.38 0 0 8.28 S 32 0 3.49 6.41 10.2 4.38 6.86 0 14.18 T 32 0 0.52 018.68 0 6.86 0 12.71 U 32 0 0.52 1.8 18.68 0.42 0 0 12.71 V 0 0 0 1.8 06.95 0 24.04 10.49 W 24 0 8.5 11.95 0 7.67 0.33 24.04 7.14 X 44 0 8.511.95 0 7.67 0.33 24.04 7.14 G 86 0 6.58 7.26 0 3.12 0 24.04 19.02 H 770 6.58 7.26 0 3.12 0 24.04 19.02 I 67 0 6.58 7.26 0 3.12 0 24.04 19.02 J57 7 6.58 7.26 0 3.12 0 7.25 11.9 K 47 17 6.58 7.26 0 3.12 0 7.25 11.9 L37 27 6.58 7.26 0 3.12 0 7.25 11.9 M 58 10 6.58 7.26 0 3.12 0 7.25 11.9Characteristics of the PMOs and their target sites listed.^(b)calculated as % of PMO target site in open structures on predictedRNA secondary structure obtained using MFOLD analysis. The position ofthe PMO target sites relative to open loops in the RNA secondarystructure is listed (0 = no ends in open loops, 1 = one end in an openloop, 2 = both ends in open loops). ^(c)In the analyses, SR bindingsites were predicted using splice sequence finder(http://www.umd.be/SSF/) software. Values above threshold are given forPMOs whose target sites cover 50% or more of potential binding sites forSF2/ASF, BRCA1, SC35, SRp40, SRp55, Tra2β and 9G8

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1. An oligomer for ameliorating DMD, the oligomer comprising at least 25contiguous bases of a base sequence selected from the group consistingof: (SEQ ID NO: 4) a) CAG XXX GCC GCX GCC CAA XGC CAX CCX GGA;(SEQ ID NO: 5) b) XXG CCG CXG CCC AAX GCC AXC CXG GAG XXC;

wherein X=U or T, wherein the oligomer's base sequence can vary from theabove sequence at up to two base positions, and wherein the molecule canbind to a target site to cause exon skipping in an exon of thedystrophin gene.
 2. The oligomer according to claim 1, wherein the exonof the dystrophin gene at which exon skipping is caused is
 45. 3. Theoligomer according to claim 1, wherein the oligomer causes an exonskipping rate of at least 50%.
 4. The oligomer according to claim 1,wherein the oligomer is between 25 and 35 bases in length.
 5. Theoligomer according to claim 1, wherein the oligomer is 30 bases inlength.
 6. The oligomer according to claim 1, wherein the oligomer isconjugated to or complexed with a distinct chemical entity.
 7. Theoligomer according to claim 1, wherein the oligomer is aphosphorodiamidate morpholino oligonucleotide (PMO).
 8. A vector forameliorating DMD, the vector encoding an oligomer according to claim 1,wherein when introduced into a human cell the oligomer is expressed . 9.A pharmaceutical composition for ameliorating DMD, the compositioncomprising an oligomer according to claim 1 or a vector according toclaim 8, and a pharmaceutically acceptable carrier, adjuvant or vehicle.10. A pharmaceutical composition according to claim 9 comprising aplurality of oligomers or vectors encoding oligomers, or a combinationof the oligomers and vectors, wherein the oligomers and/or vectors inthe pharmaceutical composition cause skipping in a plurality of exons.11. A method of ameliorating DMD in a human patient, the methodcomprising: administering a therapeutically effective amount of anoligomer according to claim 1 or a vector according to claim 8 to thepatient such that the DMD is ameliorated.
 12. The method according toclaim 11 comprising administering a therapeutically effective amount ofa plurality of oligomers to the patient, wherein the oligomers causeskipping in a plurality of exons.